Stage IV melanoma represents a significant unmet medical need. Five-year survival rates in patients with visceral metastases are in the range of 5%. While T cell immunity plays a role in the control of metastatic melanoma, passive immunotherapy is not expected to yield long-lasting immune memory. Active immunotherapy with dendritic cell (DCs)-based vaccines has the potential to induce tumor-specific effectors resulting in tumor rejection and memory T cells that might control tumor outgrowth. Our results from clinical trials testing first generation DC vaccines demonstrate: 1) Induction of melanoma-specific CD8+ T cells in patients with stage IV melanoma vaccinated with melanoma-antigen loaded DCs and 2) Induction of durable objective responses in a fraction of patients (10%) including patients who failed other therapies, provided the tumor burden was not excessive. These results provide a proof-of-principle that therapeutic immunity can be elicited. However, we need to improve the efficacy of DC vaccines. Our preliminary results demonstrate that combination of LPS and CD40 signaling generates potent DC for CD8+ T cell responses. Furthermore, LPS activation of DCs permits expansion/activation of gamma/delta T cells, a subset of innate lymphocytes not generally activated by other types of DCs. In turn, gamma delta T cells improve cross-priming by DCs. These results form the basis for our hypothesis: LPS-activated IFN-DCs will improve clinical outcomes in patients with stage IV melanoma through the activation of cytotoxic effectors cells including CD8+ T cells, NK cells, NKT cells and gamma/delta T cells. This diverse repertoire of cytotoxic effector cells will overcome Tregs. We propose four aims to address this issue. In Aim 1, we will perform a phase II clinical trial in patients with stage IV melanoma to determine whether LPS activation of IFN-DCs improves the immune and clinical response. In Aim 2, we will analyze functions of elicited melanoma-specific CD8+ T cells. In Aim 3 we will measure generation of melanoma specific CD4+ T cell immunity including antigen-specific Tregs. We will measure the expansion of NK cells, NKT cells and gamma/delta T cells in vivo in vaccinated patients for Aim 4, to determine whether the vaccine leads to activation of innate cytotoxic effector cells to exploit their potentially beneficial effect on the DC vaccine. Together, these aims will allow us to conclude whether LPS activation of IFN-DCs improves the immune and clinical efficacy of such melanoma vaccines. PUBLIC HEALTH RELEVANCE: This work will permit to develop robust and effective DC-based vaccines against melanoma. Metastatic melanoma represents an unmet medical need. Passive immunotherapy, like for example with adoptive T cell transfer, does not permit generation of specific memory T cells and is not a solution for resected high risk patients. Vaccination with DCs could become an option for patients with earlier disease stages.